Insect cells that support the replication of baculoviruses were of interest initially for the study of the basic biology of insect viruses and in agricultural use of baculoviruses for microbial pest control applications. Hink (Nature, 226:466-467, 1970) reported the first continuous insect (Lepidoptera) cells that were shown to support the replication of baculoviruses. Faulkner and Henderson (Virology, 50:920-924, 1972) demonstrated that baculoviruses could be continuously propagated in a stable insect cell line. More recently, with the development of baculovirus expression vector systems, the need for insect cells that can be used for the commercial production of human and animal health and diagnostic products has become important.
Commonly used expression systems for the production of recombinant DNA products are bacterial, yeast, insect and mammalian cells, and transgenic animals. The general method is to introduce foreign genes into the cells or organisms creating a transformed cell line or transgenic organism, which are unique for each gene product. However, in the baculovirus expression system, foreign genes are cloned into individual baculovirus vectors and a single insect cell line, susceptible to baculovirus infection, can be used to produce an unlimited number of foreign gene products.
The ideal insect cell line for use with baculovirus expression vectors would replicate continuously in suspension as single cells making them ideal for use in large-scale pharmaceutical bioreactors. The insect cells should also grow to high density with a high degree of viability in a low-cost, serum-free medium and support the replication of baculoviruses to high titers. The ideal insect cell line when infected with a genetically engineered recombinant baculovirus would produce gene products at high levels and produce those products consistently over many passages. The ideal insect cell for the production of pharmaceutical products from baculovirus expression vectors would also meet all regulatory requirements for identity and safety and be readily expandable to large-scale bioreactors for the manufacture of pharmaceutical products. Finally, due to the high cost of serum and the potential for contamination with adventitious agents such as Bovine spongiform encephalopathy, a chronic degenerative disease affecting the central nervous system of cattle (mad cow disease), the ideal insect cell line would be stored and cultured in a serum-free medium. To date, no such insect cell line with these ideal properties has been described. The current invention has as an objective to provide an insect cell line, preferably such a cell line with any or all of these ideal properties.
Baculoviruses are widely used for foreign gene expression in insect cells (see, e.g., Smith, et al., U.S. Pat. No. 4,745,051 (recombinant baculovirus) and U.S. Pat. No. 4,879,236; Summers and Smith. A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, May 1987, Texas A&M University; O'Reilly et al. Baculovirus Expression Vectors A Laboratory Manual, 1994, Oxford University Press; and references therein).
In particular, baculoviruses such as Autographa californica nuclear polyhedrosis virus (AcNPV) are grown in established Lepidoptera insect cell lines including ones derived from ovarian tissue of the fall armyworm (Spodoptera frugiperda) and the cabbage looper (Trichoplusia ni) and midgut tissue from T. ni. The cell lines in most common use to support AcNPV replication and production of recombinant products are S. frugiperda IPLB-SF-21 (Vaughn, et al. In Vitro 13:213-217, 1977) and S. frugiperda Sf-9 cells (Summers and Smith, supra), T. ni TN-368 cells (Hink, Ibid. 1970) and T. ni BTI-TN-5-B1-4 cells (Granados, U.S. Pat. Nos. 5,300,435, 5,298,418). The Sf-9 (ATCC CRL-1771) and BTI-TN-5-B1-4 (ATC, CRL 10859) cells were cloned in medium containing 10% or 8% Fetal Bovine Serum, respectively. These and other insect cells can be adapted to commercial serum-free medium, such as Sf-900 II SFM (Life Technologies, Grand Island, N.Y. 14072), using procedures known to those skilled in the art. Adapting cells repeatedly for use in the manufacture of pharmaceutical products is not desirable in that it is time consuming, may result in cells with differing properties with each adaptation, and the adapted culture of cells would contain a variable level of residual serum.
In addition, the BTI-TN-5-B1-4 cells severely clump in suspension with serum-free medium reducing its effectiveness as a host cell for foreign gene productions with baculovirus vectors. The use of non-carboxylated sulfated polyanions may help in overcoming this problem (Shuler and Dee, U.S. Pat. No. 5,728,580, Mar. 17, 1998). However, sulfated polyanions can block the infection of the cells with baculoviruses, thus complicating their use in the manufacture of recombinant DNA gene products.
Insulin is an anabolic peptide hormone important in the regulation of glucose metabolism. Insect and mammalian cells follow similar patterns of glucose metabolism from glucose to pyruvic acid; therefore it is not surprising that insulin-like peptides are produced in insects. The insect prothoracicotrophic hormone (PTTH) activates the prothoracic glands to produce the molting hormone ecdysone. The PTTH bombyxin from the silkmoth Bombyx mori has 40% homology with human insulin. Bombyxin binds to specific receptors and induces morphological changes in a B. mori cell line, specifically increasing cell size 1-2 weeks after exposure to a low concentration of bombyxin (Tanaka, M. et al., Regul. Pept. 57(3):311-318, 1995). S. frugiperda Sf9 cells have receptors for the insulin-like peptide hormone bombyxin and B. mori bombyxin binds with high affinity to receptors on S. frugiperda cells with a dissociation constant of about 0.26 nM (Fillbright, et al., Eur. J. Biochem. 245(3):774-780, 1997). Although insulin is commonly used in growth media for mammalian cells, it has not been described for use in media for insect cells. Goodwin and Adams (Ed. Kurstak, Maramorosch, Dubendorfer, Invertebrate Systems In Vitro, Elsevier/North-Holland Biomedical Press, 443-509, 1980) reported that 35 units/L of insulin did not affect the growth of Lymantria dispar insect cells. In the present invention insulin-containing serum-free medium was used in the generation of a new S. frugiperda cell line.
Reference is also made to U.S. Pat. Nos. 4,072,565, 5,135,866, 5,532,156, and 5,024,947. Inslow et al., U.S. Pat. No. 5,024,947 relates to a serum-free media for growth on insect cells and expansion of products thereby, and either individually or in any combination fails to teach or suggest the insect cell line or the methods of making or using it of the present invention. Talbot et al., U.S. Pat. Nos. 5,532,156 is directed to a hepatocyte cell line derived from the epiblast of pig blastocysts and similarly either individually or in any combination fails to teach or suggest the insect cell line or the methods of making or using it of the present invention. Heifetz et al., U.S. Pat. No. 5,135,866 provides a very low protein nutrient medium for cell culture and likewise either individually or in any combination fails to teach or suggest the insect cell line or the methods of making or using it of the present invention. And, Weiss et al., U.S. Pat. No. 4,072,565 relates to the production of viruses in tissue culture without the use of serum, and either individually or in any combination fails to teach or suggest the insect cell line or the methods of making or using it in the present invention.
Thus, it is believed that heretofore, a cell line as described and claimed herein, as well as the methods for making and using such a cell line, have not been disclosed or suggested in the art.